M ASS S PECTROMETRY The ionization source and mass analyzer are fundamental parts of a mass spectrom-

3.3.3 M ASS S PECTROMETRY The ionization source and mass analyzer are fundamental parts of a mass spectrom-

eter as production of gas-phase ions is necessary for manipulation by electric or magnetic fields. These ions are extracted into the analyzer region of the mass spectrometer where they are separated according to their mass-to-charge ratios (m=z).

3.3.3.1 Ionization Although chemical ionization (CI) cannot be used for a primary multiresidue

method, CI, and negative chemical ionization (NCI) in particular, gives better selectivity than EI for a limited number of specific compounds, which provides 58 improvements in reporting limits. NCI exhibits a high selectivity for ‘‘electron- trapping’’ compounds (e.g., halogen-containing and other heteroatomic compounds) and electron-deficient aromatic compounds. The sensitivity can be improved by two orders of magnitude compared with EI. Mass spectra produced by CI are usually dominated by ions, which, although optimum for enhancing sensitivity, offer less information in contrast with EI, which typically produces large number of charac- teristic fragments, which, when acquired under standardized condition (70 eV) can

be compared with spectra of known pesticides in published libraries such as the NIST=EPA=NIH Mass Spectral Library and Wiley Registry of Mass Spectral Data,

now available as a combined database 59 or used to generate bespoke libraries of known pesticides. 60 The identification process is based on search algorithms that

compare the spectra acquired with those of the library. A spectral match and fit factor defines the certainty of the identification.

3.3.3.2 Single Quadrupole Analyzers The quadrupole consists of four parallel metal rods. Each opposing rod pair is

connected together electrically and a radio-frequency (RF) voltage is applied between one pair of rods and the other. A direct current (DC) voltage is then superimposed on the RF voltage. Ions travel down the quadrupole in between the rods. Only ions of a certain m=z will reach the detector for a given ratio of voltages: other ions have unstable trajectories and will collide with the rods. This allows selection of a particular ion, or scanning by varying the voltages. The quadrupole can

be used in two modes: scan or single ion monitoring (SIM), also called single ion recording (SIR). In scan mode, the amplitude of the DC and RF voltages are ramped (while keeping a constant RF=DC ratio), to obtain the mass spectrum over the required mass range. Sensitivity is a function of the scanned mass range, the scan speed, and resolution. In SIM mode, the parameters (amplitude of the DC and RF voltages) are set to observe only a specific mass, or to ‘‘jump’’ between a selection of specific masses. Figure 3.1 shows a schematic of a single quadrupole instrument in SIM mode. This mode provides the highest sensitivity but users are restricted to acquiring specific ions, typically EI fragments, since more time, the dwell time, can

be spent on each mass. A longer dwell time would result in better sensitivity but, the number of data points acquired across a single peak, and the total number of pesticides that could be analyzed in a single run, are reduced. Due to insufficient

Analysis of Pesticides by Chromatographic Techniques 69

Quadrupole

Ion optics

mass filter (Q)

Detector

Ion source FIGURE 3.1 Schematic overview of a single quadrupole mass spectrometer.

sensitivity in scan mode, historically, quadrupoles have typically been operated in SIM for optimum sensitivity, limiting the amount of structural information that could

be recorded. However, such information is critical to the successful confirmation of identity of target analytes. There have been numerous reports describing the devel- opment and implementation of methods for the simultaneous determination of anything up to typically a maximum of about 400 target pesticides by GC-MS using EI with SIM and the screening for 927 ‘‘pesticides and endocrine disrupters’’ was recently reported. 61

3.3.3.3 Quadrupole Ion-Trap Analyzers Three-dimensional quadrupole ion-trap analyzers (3D QIT), 62 also termed ion-trap

detectors (ITDs), 64 have been used to carry out similar determinations. Figure 3.2 shows a schematic of a 3D QIT instrument. The principle of the trap is to store the

ions in a three-dimensional quadrupole field. Ions are removed one m=z value at a time by resonant ejection to obtain a scan recorded as a mass spectrum. Ions can be

Entrance

Exit

endcap endcap

Ions in

Ions out

Ion injection Ion ejection

Helium

Ring electrode

FIGURE 3.2 Schematic overview of a three-dimensional quadrupole ion-trap mass spec- trometer.

70 Analysis of Pesticides in Food and Environmental Samples formed internally and stored as they are formed or externally followed by injection

and storage. The ability to selectively store ions provides a substantial improvement in sensitivity when compared with a quadrupole instrument when recording mass spectra and so permits the recording of complete mass spectra in concentration ranges in which quadrupole instruments have historically had to operate in SIM mode. This aspect is particularly advantageous for pesticide residue analysis when the components under investigation are present in the sample only in very small concentrations and an unambiguous identification is required. Moreover, no selec- tion of characteristic ions is necessary during data acquisition for MS with a 3D QIT, permitting investigation of unknown samples, that is, screening of samples on the basis of complete mass spectra. Thus, substances that were not originally sought can

be detected by revisiting the data. This is not possible in the case of the SIM technique, which operates on the principle of the selection of previously known substance and characteristic ions.

Disadvantages of GC-MS using original 3D QITs were related to space charge problems, leading to lower mass resolution and mass shifts 65 and ion=molecule

reactions called ‘‘self-CI.’’ 66 Although modern instruments have various techniques to prevent overfilling of the trap, this can still be a problem when analyzing

pesticides at low levels in dirty matrices because the trap is filled with ions derived from matrix leaving little space for the small number of analyte ions. The limited storage of ions has also limited the dynamic range of the 3D QIT.

3.3.3.4 Tandem Mass Spectrometry Analyzers As both single quadrupole and 3D QIT work at unit mass resolution, selectivity is

limited, so these instruments can suffer from reduced sensitivity due to the contri- bution to the analyte signal from chemical noise. Although low reporting limits might be possible for simple matrices using GC-MS, these instruments can provide insufficient selectivity for complex food matrices. Tandem mass spectrometry

(MS=MS), 67 in which mass-selected ions are subjected to a second mass spectro- metric analysis, can provide increased selectivity, which reduces the contribution to the analyte signal from isobaric interference leading to improvements in sensitiv-

ity. 68 Hence, lower limits of detection become achievable when using GC-MS=MS for pesticide residue analysis in complex matrices. The same selectivity, achieved by monitoring the transition from one parent ion to a characteristic product ion, provides

a greater degree of confidence for confirmation of identity than SIM, which can suffer from isobaric interferences. Based on the current EU quality control proced- ures for pesticide residue analysis, 19 if using GC-MS, four ions have to be detected

and all ion ratios have to be within the specified tolerance intervals for identity to be confirmed. Additional legislation directed at residues of substances in live animals and animal products introduced an identification point (IP) system that was weighted 69 to the selectivity of the method used. When using the more selective MS=MS technique, monitoring and detection of two transitions exhibiting a ratio within tolerance is sufficient, as the precursor earns 1 point and each product ion earns

1.5 points, 4 points in total. The IP system has been applied to the determination of pesticide residues in animal products 25 and may find wider usage.

Analysis of Pesticides by Chromatographic Techniques 71 The capability of the 3D QIT to store ions of a single m=z value to the exclusion

of ions of all other m=z values allows for MS=MS by means of collision-induced dissociation (CID) within a single mass analyzer. 70 An ion can be stored as a precursor, and that stored ion can then be manipulated to collide with the cooling gas molecules to produce product ions. By ramping the RF voltage, or by applying supplementary voltages on the end cap electrodes, or by combination of both, it is possible to keep only one ion in the trap, fragment it by inducing vibrations, and observe the fragments as they are sequentially ejected from the trap. The high efficiency for ion-trap MS=MS results from the parent and product ions remaining in a single ion trap and not transported from one chamber to another, eliminating transport losses. The application of wideband excitation (activation) and normalized collision energy leads to highly reproducible mass spectra. Hence, the main advan- tage of using a 3D QIT for GC-MS=MS is that full product ion spectra can be

generated from trace amounts of pesticides for comparison with MS=MS libraries. 71 The performance of tandem quadrupole GC-MS=MS has long been recog-

nized, 72 but the price has been out of the reach of many laboratories involved in pesticide residue analysis that would benefit from this technology. With the recent

introduction of GC-MS=MS instruments, based on the tandem quadrupole technol- ogy of existing LC-MS=MS platforms, the number of laboratories using it for pesticide residue analysis is growing. The analyzer of a triple quadrupole instrument

consists in two quadrupoles, separated by a collision cell. 73 The first quadrupole is used in SIM mode to select a first ion (precursor), which is fragmented in the

collision cell. This is typically achieved in the collision cell by accelerating the ions in the presence of a collision gas. The energy of the collision with the gas can be varied to allow different degrees of fragmentation. The resulting fragments are analyzed by the second quadrupole and also typically used in SIM mode to monitor a specific fragment (product), the process known as multiple reaction monitoring (MRM) (also called selected reaction monitoring, SRM). Figure 3.3 shows a schematic of a triple quadrupole instrument in MRM mode. As two analyzers increase the selectivity, the ion signal is reduced during the transmission, but the chemical noise, which is a major limitation for complex samples, is also largely decreased, leading to an improvement of the signal-to-noise ratio.

One limitation in GC-MS=MS, on either type of instrument, arises from the fragmentation provided by EI as often the total ion current is spread on many

Ion optics Quadrupole

Collision cell

Quadrupole

mass filter (Q) quadrupole (q) mass filter (Q)

Detector

Ion source FIGURE 3.3 Schematic overview of a triple quadrupole mass spectrometer.

72 Analysis of Pesticides in Food and Environmental Samples fragments, resulting in low intensity of ions that can be selected as parent ions for

MS=MS experiments. The primary advantage of 3D QIT is that multiple MS=MS experiments can be performed quickly without having multiple analyzers. Hence, the introduction of MS=MS on a 3D QIT was a major breakthrough as it brought down

the cost of tandem mass spectrometry. 74 Unlike MRM using a triple quadrupole, however, MS=MS using 3D QIT is restricted to concurrent acquisition of a limited number of precursor ions.

3.3.3.5 Time-of-Flight Analyzers The use of TOF technology 75 provides an innovative approach to overcoming the

drawbacks that limit the exploitation of mass spectrometers for detecting pesticides at trace levels while retaining full spectral information as a tool for confirmation of 76 identity. The design of the orthogonal acceleration (oa) TOF, into which pulses of ions are extracted orthogonally from a continuous ion beam, the availability of fast-recording electronics, together with improvements in signal deconvolution techniques, were major breakthroughs in the development of modern GC-TOF instruments.

Figure 3.4 shows a schematic of a TOF instrument. As the name implies, separation of ions in a TOF mass analyzer is accomplished by measuring their flight time in a field-free tube based on the fact that ion velocity is mass-dependent. The ions generated in an EI source are initially accelerated to get discrete packages with a

constant kinetic energy, which are ejected into the mass analyzer using pulsed electric field gradient oriented orthogonally to the ion beam. Reflectrons (ion mir- rors) are used to compensate for variations in initial energy distribution. Ions are reflected based on their forward kinetic energy. The more energetic the ion, the deeper it penetrates the retarding field of the reflection before getting reflected. This allows an energetic ion, traveling a longer flight path, to arrive at the detector at the

Orthogonal accelerator

Drift region

Reflectron Detector

Detector

Ion source

FIGURE 3.4 Schematic overview of a TOF mass spectrometer.

Analysis of Pesticides by Chromatographic Techniques 73 same time as the less energetic ions of the same mass. Ions are detected using a

multichannel plate detector (MCP) and detection of ‘‘ion events’’ converted from an analog signal to a digital record. Flight times, which are proportional to the square root of the m=z value of an ion, are in the order of microseconds. Consequently, TOF MS can operate at very high repetition rates and between 20 and 500 spectra per second can be stored. The effort to exploit these unique features has resulted in the development of two types of commercial spectrometers differing in their basic characteristics: instruments using unit-resolution instruments that feature a high acquisition speed and elevated resolution analyzers with only moderate acquisition speed. The application potential of these approaches is obviously complemen- tary. 77,78 Both approaches are characterized by high sensitivity due to improved

mass analyzer efficiency and continuous acquisition of full range mass spectra. Mass analyzer efficiency of the TOF-MS instruments is as high as 25% in full spectra acquisition (quadrupole 0.1%). Generation of complete mass spectra from residues of pesticides even at trace levels enables searching against library reference spectra for identification. The fast repetition rate ensures that no changes in the ratios of analyte ions across the peak occur during the acquisition of the mass spectrum and, consequently, no spectral skew, which is commonly observed by scanning instru- ments, is encountered. The generation of full range mass spectra also provides the ability to review archived data for new compounds outside the scope of the initial analysis. For example, in response to an EU Rapid Alert in late December 2006, those laboratories within the EU using GC-TOF MS were able to look back again at historical data and identify residues of isofenphos-methyl even though no routine government testing program in any EU member state had included tests for iso- fenphos-methyl at that time.

High-speed TOFs, operating at unit mass resolution but with very fast scan rates (e.g., 500 spectra=s), can provide the data density necessary to accurately define

narrow chromatographic peaks typical for fast and ultrafast GC 79 and GC 3 GC. 80 The high data acquisition rate and the absence of any spectral skew allow overlap- ping signals to be automatically deconvoluted based on their mass spectra. 81 The GC-TOF instruments with elevated resolution (typically 5000 –7000 FWHM) and good mass accuracy (e.g., 5 ppm RMS) have so far had a more limited application to

the analysis of pesticide residues. 82 The elevated resolution and good mass accuracy can be significantly used to reduce the contribution from isobaric interference by evaluating data with a narrow mass window (typically 50 mDa), which improves detectability of the analytes. Measurement of an accurate mass of a particular ion also provides additional information for confirmation of identity for target compound analysis and, more importantly, aids the assignment of unknown compounds based on calculation of their elemental composition. High mass accuracy is attainable by using a lock mass calibration procedure for which a reference compound is continu- ously supplied into the ion source during analysis. On the basis of a previously performed mass calibration over a given mass range and a defined exact mass of the reference ion (the lock mass), the values of all masses in the acquired spectra are

automatic and continually corrected. Mass accuracy varies with ion intensity. Care is required when applying exact mass windows as if the window is set too narrow, peaks may be underestimated or missed altogether. It is possible to search for

74 Analysis of Pesticides in Food and Environmental Samples compounds of interest by measurement of exact mass and isotope pattern of fragment

ions and compare with the accurate masses in the reference spectra. Using this approach, the identification of the analyte can be based not only on retention time and EI mass spectrum, but thanks to the exact mass measurement also on elemental composition. In addition, the measurement of exact mass may aid the identification of unknown compounds through the calculation of elemental composition. In prac- tice, this is a very demanding task as EI spectra rarely exhibit a molecular ion so a good knowledge of the fragmentation mechanism is required.

The main disadvantage of the TOF as an analyzer for quantitative GC-MS is the limited linear dynamic range compared with conventional MS instrumentation. The analog signal from the ion detector is converted to a digital record by a fast analog- to-digital (ADC)-based continuous averager, also called an integrating transient recorder (ITR). With an ITR, the ion rates can be increased so that many ions of the same m=z value arrive at the ion detector simultaneously. The result is an analog voltage pulse whose amplitude is approximately proportional to the number of ions in the pulse. An ADC samples the output waveform of the ion detector and periodically converts the measured analog voltage to a digital representation, which is sequentially stored in a digital memory to form a single record. Although the linear dynamic range of the ITR is limited to two orders of magnitude, it has been expanded to approximately four orders of magnitude by application of continuing improvements in both hardware and software features.

While GC-MS, using EI with SIM on a single quadrupole, still provides the widest scope for pesticide residue analysis, GC-MS=MS and GC-TOF MS offer two unique solutions. However, both are still used by a few laboratories. Although initial purchase cost remains higher than conventional benchtop instruments, which may currently prohibit use within routine laboratories, both approaches offer considerable benefits and so usage is likely to grow.